Transplanted Neocortical Neurons Migrate to Repopulate Selectively Neuron‐Deficient Regions After Photolytic Pyramidal Neuron Degeneration

The purpose of these experiments was to study neuronal migration and cellular integration following transplantation of immature neocortical neurons or pluripotent transformed neuronal precursor cell lines into a "customgenerated" pyramidal neuron deficient host. The experiments investigated both specificity of laminar positioning during cerebral development, and control mechanisms that may be manipulated for potential future neocortical transplantation. A novel model of selective neocortical degeneration using non-invasive laser illumination/1/was used to provide injury to callosally projecting pyramidal neurons that is geographically defined, slowly progressive, and cell-type specific. This selective neuronal injury allows precise control over the host anatomical substrate for cellular transplantation. Unfocused laser energy, at long wavelengths which penetrate through tissue without major absorption, can cause extremely selective, non-invasive damage to desired subpopulations of neurons targeted by retrograde incorporation of cytolytic chromophores which are activated by the laser energy. Intermixed neurons, glia, axons, blood vessels, and connective tissue remain intact. Selective degeneration was effected to pyramidal neurons within neocortical lamina II/III of neonatal and young adult mice (see Figure, panel A). Embryonic day 14 or 17 (E14, E17) neocortical cell suspensions, containing recently postmitotic neurons destined to form lamina V and VI or II/III respectively, were transplanted adjacent to these geographically defined regions of ongoing neuron degeneration. Cellular injections spanned lamina II to V, to provide donor neurons with both lateral and laminar choice for possible migration and integration. Donor cells were labeled in vitro with various combinations of unique fluorescent and electron-dense nanospheres, tritiated thymidine, and DiI, allowing distinct identification of donor cells at both lightand electron-microscopic levels. All transplanted cell suspensions were followed in tissue culture to assess labeling, viability, and differentiation following the cell preparation and stereotaxic transplantation via pulled glass micropipette. In one set of experiments, lamina II/III neuronal degeneration was effected, E17 neurons were transplanted (neuronal birthdate destined to form these superficial layers), and experimental and control cortices were examined 1 day to 12 weeks later. Camera lucida reconstructions and higher magnification neuronal identification (by unique intracellular fluorescence within morphologically neuronal cells) were used to assess donor neuron migration and morphologic differentiation; EM was used to further confirm donor identities of migrated neurons (by incorporation of both electron dense nanospheres and a colloidal gold second label prior to transplantation). Neurons in experimental cortices migrated up to 1 mm and integrated specifically within the neurondeficient zones. Migration and integration did not occur in normal, unaffected deeper layers IV to VI of these experimental mice, nor in the normal lamina II/III bordering the transplantation site on the side opposite the neurondeficient region. Control grafts into intact hosts, grafts into kainic acid lesioned cortices, grafts of hypo-osmotically lysed neocortical cells, and grafts of cerebellar cell suspensions revealed